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. 2017 Jan 10;78(12):1847-1854.
doi: 10.1292/jvms.16-0342. Epub 2016 Sep 15.

Development and application of multiplex PCR assays for detection of virus-induced respiratory disease complex in dogs

Chutchai Piewbang  1 , Anudep Rungsipipat, Yong Poovorawan, Somporn Techangamsuwan
Affiliations

Development and application of multiplex PCR assays for detection of virus-induced respiratory disease complex in dogs

Chutchai Piewbang et al. J Vet Med Sci. .

Abstract

Canine infectious respiratory disease complex (CIRDC) viruses have been detected in dogs with respiratory illness. Canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), canine adenovirus type 2 (CAdV-2) and canine herpesvirus 1 (CaHV-1), are all associated with the CIRDC. To allow diagnosis, two conventional multiplex polymerase chain reactions (PCR) were developed to simultaneously identify four RNA and two DNA viruses associated with CIRDC. The two multiplex PCR assays were then validated on 102 respiratory samples collected from 51 dogs with respiratory illness by sensitivity and specificity determination in comparison to conventional simplex PCR and a rapid three-antigen test kit. All six viruses were detected in either individual or multiple infections. The developed multiplex PCR assays had a >87% sensitivity and 100% specificity compared to their simplex counterpart. Compared to the three-antigen test kit, the multiplex PCR assays yielded 100% sensitivity and more than 83% specificity for detection of CAdV-2 and CDV, but not for CIV. Therefore, the developed multiplex PCR modalities were able to simultaneously diagnose a panel of CIRDC viruses and facilitated specimen collection through being suitable for use of nasal or oral samples.

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Figures

Fig. 1.
Fig. 1.
Analytical sensitivity test of the (A–D) simplex and (E) multiplex RT-PCR of RNA-associated CIRDC viruses. (A) CIV, (B) CPIV, (C) CDV and (D) CRCoV. Two-fold serial dilutions of the positive controls ranging from 20 −2−10ng/reaction were assayed. Detection threshold was equal in both the simplex and multiplex modalities and revealed minimal detectable dilution at 2−6(CIV), 2−5 (CPIV) and ≥2−10 (CDV and CRCoV) ng/reaction. M=DNA marker 100 bp, −ve=negative control.
Fig. 2.
Fig. 2.
Analytical sensitivity test of (A, B) simplex and (C) multiplex PCR of DNA-associated CIRDC viruses. (A) CAdV-2 and (B) CaHV-1. Two-fold serial dilutions from 20 −2−10ng/reaction were tested. Detection threshold was similar in both the simplex and multiplex modalities and revealed minimal detectable dilution at 2−4 (CAdV-2) and 2−6 (CaHV-1)ng/reaction. M=DNA marker 100 bp, −ve=negative control.
Fig. 3.
Fig. 3.
Results of the (A) multiplex RT-PCR and (B) multiplex PCR tested on clinical samples (S1–S14). M=DNA marker 100 bp, −ve=negative control, +ve=positive control.
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